Sergio Giunta

Inflammaging and anti-inflammaging: A systemic perspective on aging and longevity emerged from studies in humans

A large part of the aging phenotype, including immunosenescence, is explained by an imbalance between inflammatory and anti-inflammatory networks, which results in the low grade chronic pro-inflammatory status we proposed to call inflammaging. Within this perspective, healthy aging and longevity are likely the result not only of a lower propensity to mount inflammatory responses but also of efficient anti-inflammatory networks, which in normal aging fail to fully neutralize the inflammatory processes consequent to the lifelong antigenic burden and exposure to damaging agents. Such a global imbalance can be a major driving force for frailty and common age-related pathologies, and should be addressed and studied within an evolutionary-based systems biology perspective. Evidence in favor of this conceptualization largely derives from studies in humans. We thus propose that inflammaging can be flanked by anti-inflammaging as major determinants not only of immunosenescence but eventually of global aging and longevity.

Do men and women follow different trajectories to reach extreme longevity

Gender accounts for important differences in the incidence and prevalence of a variety of age-related diseases. Considering people of far advanced age, demographic data document a clear-cut prevalence of females compared to males, suggesting that sex-specific mortality rates follow different trajectories during aging. In the present investigation, we report data from a nationwide study on Italian centenarians (a total of 1162 subjects), and from two studies on centenarians living in two distinct zones of Italy, i.e., the island of Sardinia (a total of 222 subjects) and the Mantova province (Northern Italy) (a total of 43 subjects). The female/male ratio was about 2:1 in Sardinia, 4:1 in the whole of Italy, and about 7:1 in the Mantova province. Thus, a complex interaction of environmental, historical and genetic factors, differently characterizing the various parts of Italy, likely plays an important role in determining the gender-specific probability of achieving longevity. Gender differences in the health status of centenarians are also reported, and an innovative score method to classify long-lived people in different health categories, according to clinical and functional parameters, is proposed. Our data indicate that not only is this selected group of people, as a whole, highly heterogeneous, but also that a marked gender difference exists, since male centenarians are less heterogeneous and more healthy than female centenarians. Immunological factors regarding the age-related increase in pro-inflammatory status, and the frequency of HLA ancestral haplotypes also show gender differences that likely contribute to the different strategies that men and women seem to follow to achieve longevity. Concerning the different impact of genetic factors on the probability of reaching the extreme limits of the human life-span, emerging evidence (regarding mtDNA haplogroups, Thyrosine Hydroxilase, and IL-6 genes) suggests that female longevity is less dependent on genetics than male longevity, and that female centenarians likely exploited a healthier life-style and more favorable environmental conditions, owing to gender-specific cultural and anthropological characteristics of the Italian society in the last 100 years.

In vitro peroxidase oxidation induces stable dimers of β-amyloid (1-42) through dityrosine bridge formation

beta-amyloid (A beta) is a normal soluble peptide found in the cerebrospinal fluid (CSF) and other biological fluids. A beta fibrils are associated with Alzheimers disease (AD) senile plaques. We have used purified soluble A beta (1-42) and A beta (12-28) peptides in order to determine the oxidative modification induced in these peptides by exposure to peroxidase and hydrogen peroxide. We have demonstrated that under these in vitro conditions, dimeric forms of A beta (1-42) can be detected by high-resolution polyacrylamide SDS-PAGE electrophoresis. Further experiments performed by reverse-phase high performance liquid chromatography (RP-HPLC), and monitored by fluorescence detection, showed that the dimeric A beta (1-42) forms induced by the peroxidase reaction are the outcomes of dityrosine bridge formation. This cross-link results from the enzyme catalyzed oxidation. During this reaction, phenolic coupling of tyrosine residues of two A beta (1-42) peptides occurs. No detectable peroxidative modifications were observed with the A beta (12-28) peptide which lacks a tyrosine residue. Since oxidative stress is thought to be associated with AD, the experimental model described here can help in understanding the early events leading to chemical, structural and conformational modifications before the conversion of sA beta to amyloid fibrils and eventually the formation of senile plaques in AD.

Albumin protects human red blood cells against Aβ25-35-induced lysis more effectively than ApoE

Inhibition of the lysis of human red blood cells (RBCs) exposed to amyloid peptide A&bgr;25–35 is an in vitro model for screening natural and synthetic substances potentially protective against amyloid damage. In this system, human serum and a component, namely apolipoprotein E (apoE), completely prevent RBC lysis. This report demonstrates that albumin, another serum component, is 8-fold more protective: a concentration of 12.5 μg/ml protects RBCs against 20 μM-A&bgr;25–35, and prevents the formation of fibrillar A&bgr;25–35 aggregates stainable by Congo Red. The biological relevance of these findings is suggested by the following: (1) a large fraction (∼90%) of circulating A&bgr;1–42 is bound to albumin; (2) albumin immunoreactivity is present in brain amyloid plaques; and (3) incubation of A&bgr; with albumin rapidly decreases detectable levels of free A&bgr; suggesting epitope masking. The results add new and important functional consequences to the amyloid-albumin relationship and imply that experimental systems investigating A&bgr; cytotoxicity should consider the protective interaction of albumin.

Amiloride, a diuretic with in vitro antimicrobial activity.

The effect of amiloride, an inhibitor of passive sodium influx in animal cells, was investigated on the in vitro bacterial growth. Amiloride blocked the growth of different bacterial strains at concentrations ranging from 25 to 1,300 micrograms/ml. While generally the block was bacteriostatic and bacteria, on amiloride removal, recovered their ability to growth, the drug showed a killing activity on hemolytic streptococci. Gram-positive bacteria revealed a greater susceptibility to amiloride than gram-negative ones. Although an hitherto unknown effect of amiloride cannot be excluded, from the known mechanism of action of amiloride on animal cells it might be suggested that sodium permeability plays a critical role on bacterial multiplication.

Differential course of HIV-1 infection and APOE polymorphism

Burt et al. (1) demonstrate that the APOE e4/e4 and other e2+ or e4+ genotypes accelerate HIV mortality, a gene dosage effect of the e4 allele on steady-state viral load, and they associate e4 with enhanced HIV-1 cell entry. They did not find an excess of HIV-associated dementia (HAD) among e4+ patients.

In vitro block of murine L 1210 leukemia cell growth by amiloride, an inhibitor of passive Na+ influx

The present study was aimed to decide whether Na+ influx can be involved in regulation of murine L 1210 leukemia cell growth. Cells were cultivated in the presence of different concentrations of amiloride and cellular growth was monitored by 3H-thymidine incorporation/10(5) cells. This drug inhibited cell growth in concentrations ranging from 1 X 10(-5) to 1 X 10(-3) mmol/ml. Even short time treatments with amiloride caused irreversible alterations: the cells, although survived, lost their ability to divide. The results support the hypothesis that Na+ influx is necessary for the duplication of tumor cells.

Differential course of HIV-1 infection and apolipoprotein E polymorphism

We studied the course of infection with human immunodeficiency virus type 1 (HIV-1) in relation to apolipoprotein E (APOE) polymorphism found for 209 Italians treated at Infectious Disease Clinics in Rome and Modena. Clinically, patients were classified into four groups according to the yearly rate of decline in CD4+ cell count (LTNP: long-term non-progression; SLOW, ’NORMAL’ or RAPID). Patients at both extremes of the clinical spectrum, i.e. those who rapidly progressed to AIDS and those with stable high CD4 cell counts, had few APOE ɛ4 and ɛ2 alleles (P = 0.04). Detailed clinical information was then used to construct four model-based clinical profiles using grade-of-membership analysis (GoM), predictive of APOE genotypic frequencies: 1. The clinical profile associated with good long-term prognosis lacked ɛ2 (P=0.01); 2. Disease progression to AIDS was associated with ɛ4 and ɛ2, most evident for zidovudine-lamivudine regimens without a protease inhibitor (P = 0.03); and, 3. AIDS patients had low ɛ4 and ɛ2 frequencies, consistent with a high mortality rate among ɛ4+ and ɛ2+ AIDS patients. These findings suggest allele-specific immunomodulatory effects involving inherited APOE isoform important enough to alter the clinical course of HIV infection and, possibly, drug efficacy. They imply a connection between lipid metabolism and immunity potentially relevant to common disorders.

In vitro apolipoprotein E protects human red blood cells against lysis induced by amyloid-beta (Aβ) fragment 25-35

Mattson et al.9 demonstrated lysis of human red blood cells (RBC) exposed to amyloid peptide Aβ25-35′ a new experimental model for amyloid-beta toxicity. Lysis resulted from pore formation in the RBC membranes and was completely prevented by concurrent exposure to Congo red. We demonstrate that human serum, purified ApoE from human plasma, and recombinant isoforms of ApoE neutralize the Aβ25-35 cytotoxicity: the E2 and E4 isoforms were marginally more effective than E3. Second, we demonstrate that Aβ25-35 forms fibrils in the reaction mixtures using electronmicroscopy. Together these results suggest that the RBC model might be useful in preliminary identification of natural and synthetic substances able to protect against amyloid-beta cytotoxic effects due to fibrillar Aβ25-35. Such compounds would be candidate molecules for testing in neuronal systems.

β-amyloid fragment 25-35 induces changes in cytosolic free calcium in human platelets

The beta-amyloid (βA) peptide has a central role in Alzheimer’s disease (AD). Indeed, the major histopathologic hallmarks of AD include βA deposits in brain parenchima (senile plaques) and also around and within the walls of blood vessels (cerebral amyloid angiopathy).1 βA deposits are the final result of an amyloidogenic process triggered by oxidative and conformational modifications and by crosslinking of βA.2–4 βA is a peptide produced by the proteolysis of the amyloid precursor protein (APP). It has been reported that abnormalities of brain APP metabolism may be reflected in platelets, which also possess all the machinery to generate the amyloid β (Aβ)-fragment from APP; moreover, platelets are the primary source of Aβ-peptide in human blood.5–7 In human cortical neurons β-amyloid peptides have been shown to destabilize calcium homeostasis and cause neurodegenerative effects.8,9 Alteration in calcium homeostasis induced by Aβ have been reported also for nonneuronal cells.10,11 Moreover, it has been demonstrated that Aβ25–35 increases cellular APP by inhibiting its secretory processing in human extraneuronal cells.12 The data reported above encourage further exploration about amyloid and platelets as a peripheral laboratory mirroring central amyloid metabolism and activity.1 In the present paper we investigate the effects of neurotoxic Aβ25–35 peptide on platelets and we show that this Aβ-fragment is able to induce dosedependent changes of calcium concentration and degenerative effects in normal human platelets. Blood samples were taken from male healthy donors aged between 31 and 50 years (mean age: 38 ± 7). Citrated blood was centrifuged for 10 min at 200 × g to obtain platelet-rich plasma (PRP). Platelets were separated from PRP by centrifugation at 2000 × g for 20 min and washed twice in phosphate-buffered saline (PBS) 0.1 M, pH 7.4. Platelets were resuspended at a density of 108/ml in Hepes buffer containing 145 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM Hepes, 10 mM glucose, adjusted to pH 7.4. Ten μM prostaglandin E1 (PGE1) was added to prevent aggregation. The platelet suspension was incubated in 1 μM Aβ25–35